Microbe Profile: Wigglesworthia glossinidia: the tsetse fly’s significant other

Wigglesworthia glossinidia is an obligate, maternally transmitted endosymbiont of tsetse flies. The ancient association between these two organisms accounts for many of their unique physiological adaptations. Similar to other obligate mutualists, Wigglesworthia ’s genome is dramatically reduced in size, yet it has retained the capacity to produce many B-vitamins that are found at inadequate quantities in the fly’s vertebrate blood-specific diet. These Wigglesworthia -derived B-vitamins play essential nutritional roles to maintain tsetse’s physiological homeostasis as well as that of other members of the fly’s microbiota. In addition to its nutritional role, Wigglesworthia contributes towards the development of tsetse’s immune system during the larval period. Tsetse produce amidases that degrade symbiotic peptidoglycans and prevent activation of antimicrobial responses that can damage Wigglesworthia . These amidases in turn exhibit antiparasitic activity and decrease tsetse’s ability to be colonized with parasitic trypanosomes, which reduce host fitness. Thus, the Wigglesworthia symbiosis represents a fine-tuned association in which both partners actively contribute towards achieving optimal fitness outcomes.


Graphical abstract
Wigglesworthia (represented as blue rods) are obligate endosymbionts found exclusively within tsetse flies.The bacterium resides within bacteriocytes that collectively form a bacteriome organ attached to the anterior end of the tsetse midgut.This population of Wigglesworthia produces several B-vitamins that are found at metabolically inadequate quantities in tsetse's vertebrate blood-specific diet.An additional population of the bacterium resides extracellularly within maternal milk gland secretions.These Wigglesworthia are vertically transmitted to developing intrauterine larvae and produce molecules that trigger maturation of the fly's immune system.Wigglesworthia residing within the bacteriome organ produce B-vitamins to supplement tsetse's vertebrate blood diet.Wigglesworthia present in milk secretions are vertically transmitted to larvae and boost immune system maturation.

Abstract
Wigglesworthia glossinidia is an obligate, maternally transmitted endosymbiont of tsetse flies.The ancient association between these two organisms accounts for many of their unique physiological adaptations.Similar to other obligate mutualists, Wigglesworthia's genome is dramatically reduced in size, yet it has retained the capacity to produce many B-vitamins that are found at inadequate quantities in the fly's vertebrate blood-specific diet.These Wigglesworthia-derived B-vitamins play essential nutritional roles to maintain tsetse's physiological homeostasis as well as that of other members of the fly's microbiota.In addition to its nutritional role, Wigglesworthia contributes towards the development of tsetse's immune system during the larval period.Tsetse produce amidases that degrade symbiotic peptidoglycans and prevent activation of antimicrobial responses that can damage Wigglesworthia.These amidases in turn exhibit antiparasitic activity and decrease tsetse's ability to be colonized with parasitic trypanosomes, which reduce host fitness.Thus, the Wigglesworthia symbiosis represents a fine-tuned association in which both partners actively contribute towards achieving optimal fitness outcomes.

TAXONOMY
Phylum: Proteobacteria; Class: Gammaproteobacteria; Order: Enterobacterales; Family: Erwiniaceae; Genus: Wigglesworthia; Species: Wigglesworthia glossinidia.The species name is used for this clade of tsetse fly symbionts with host species appended to designate strains.The Wigglesworthia type strain was described in 1995 within Glossina morsitans morsitans [1].The name is in recognition of insect physiologist Sir Vincent Brian Wigglesworth F.R.S. (1899-1994), who described the Wigglesworthia-tsetse symbiosis in 1929.

PROPERTIES
Tsetse's obligate endosymbiont Wigglesworthia presents two membrane bilayers, is rod-shaped and is approximately 5 µm in length.Wigglesworthia reside freely within the cytoplasm of specialized epithelial cells, known as bacteriocytes, which collectively form the bacteriome organ that is attached to tsetse's anterior midgut.This population of Wigglesworthia provide their tsetse host with essential nutrients, including B-vitamins, that are lacking in physiologically sufficient quantities in the fly's strict vertebrate blood-specific diet.A second population of Wigglesworthia resides extracellularly within the accessory (milk) gland tubules of females.Vertical transmission of Wigglesworthia occurs via milk gland secretions that seed the bacterium to the developing intrauterine larva.

GENOME
The genomes of Wigglesworthia glossinidia 'morsitans' and 'brevipalpis' have been fully sequenced and annotated [2,3].These genomes demonstrate high gene conservation and chromosomal synteny and have a median size of 0.7 Mb, 623 proteins and 23.8 % G+C nucleotide content.The genome contains two rRNA operons with contiguity between rRNA genes.The small genome size reflects the relaxed evolutionary selection on less relevant genes in this specialized lifestyle and their subsequent loss, and higher levels of genetic drift due to population bottlenecks during transmission.The genome is polyploid within individual Wigglesworthia cells.Notably, Wigglesworthia genomes lack the dnaA gene and oriC region that encode the DNA replication initiation protein and the origin of replication, respectively, probably indicating alternative modes of regulation.The retention of flagellum synthesis capabilities may confer motility to extracellular Wigglesworthia during vertical transmission and aid in the colonization of larval bacteriomes.A small cryptic plasmid (~5 kb) is retained by Wigglesworthia, suggesting functional relevancy towards symbiont and/or tsetse biology.

PHYLOGENY
Based on 16S rRNA gene sequences, the genus Wigglesworthia forms a distinct lineage in the Gamma subdivision of Proteobacteria and in congruence with tsetse species phylogeny.These parallel phylogenies support an evolutionary scenario of an initial Wigglesworthia infection prior to tsetse species radiation and subsequent co-diversification of symbiont and tsetse host [4].

KEY FEATURES AND DISCOVERIES
Wigglesworthia contributions towards tsetse nutrition: Wigglesworthia's extensive and intimate co-evolution with the tsetse fly is reflected by the bacterium's crucial metabolic contributions to the maintenance of tsetse's fitness as well as the fitness of the fly's other microbial partners.For example, Wigglesworthia is intimately tied to tsetse's reproductive success.Pregnant female tsetse utilize proline as a prominent haemolymph-borne energy source to fuel the production of milk that serves as the sole nutrient source for developing intrauterine larvae.Vertebrate blood does not contain sufficient quantities of proline, and as such, female flies recycle alanine, which is a byproduct of the tricarboxylic acid cycle, back into proline.This process is facilitated by the enzyme alanine-glyoxylate aminotransferase, which requires Wigglesworthia-derived vitamin B6 (pyridoxal phosphate) as a co-factor.When pregnant female tsetse flies are treated with antibiotics to remove their Wigglesworthia, milk production ceases and larval offspring perish [5].Wigglesworthia also produce vitamin B9 (folate), and experimental inhibition of folate production (via blood meal supplementation with glyphosate) prolongs tsetse blood meal digestion and the development of intrauterine larval and free-living pupal stages.Additionally, newly emerged adults derived from glyphosate-treated mothers present significantly smaller wings than do their counterparts derived from wild-type mothers [5].
Tsetse not the only benefactor of Wigglesworthia-produced vitamins.African trypanosomes, which are transmitted by tsetse and cause socioeconomically devastating human and animal African trypanosomiases, are also folate auxotrophs that, like their fly host, rely on Wigglesworthia to provide this vitamin.Accordingly, inhibition of Wigglesworthia folate production reduces tsetse's competence as a vector of pathogenic African trypanosomes [6].Many tsetse flies also house the facultative endosymbiont Sodalis.This bacterium lacks the genetic machinery to produce vitamin B1 (thiamine), which it relies on for proper growth.Wigglesworthia produces thiamine, and Sodalis probably scavenges this vitamin from its environment via the activity of a thiamine ABC transporter [5].The Wigglesworthia metabolic contributions described above provide evidence of how the obligate bacterium enhances tsetse's fitness as well as the fitness of members of the fly's microbial community.
Wigglesworthia contributions towards tsetse immunity: In addition to providing essential vitamins to the tsetse holobiont, Wigglesworthia is intimately associated with the development and proper function of the fly's immune system.Tsetse offspring that undergo their entire developmental programme in the absence of Wigglesworthia present several immunocompromised phenotypes during adulthood.For example, Wigglesworthia-free flies fail to produce a structurally complete peritrophic matrix, which is a chitinous sleeve that lines the fly's midgut and serves as a barrier to establishment of trypanosome infection.This phenotype contributes to the establishment and progression of trypanosome infections in tsetse's midgut [7].Wigglesworthia-free tsetse adults also present a highly depleted population of sessile and circulating haemocytes.These immune cells phagocytose foreign invaders and produce melanin that encapsulates pathogens and forms clots at cuticular wound sites [8].These findings indicate that Wigglesworthia plays a crucial role in tsetse's competence as a vector of trypanosomes as well as the fly's ability to recover from injuries.
Wigglesworthia-tsetse dialogue: Gene expression analysis of the bacteriome organ reveals a closely tailored dialogue between tsetse and Wigglesworthia.Bacteriocytes produce an abundant peptidoglycan recognition protein LB (PGRP-LB) that has amidase activity to degrade symbiotic peptidoglycan, which is a major elicitor of host antimicrobial immune responses that can otherwise damage the bacteria [9], and a multivitamin transporter (smvt) that probably aids in the dissemination of nutrients provisioned by Wigglesworthia [10].The Wigglesworthia-responsive PGRP-LB also has anti-parasitic activity and decreases tsetse's ability to be colonized with parasitic trypanosomes, which in infected insects reduce host fitness [9].Wigglesworthia overproduces molecular chaperones (GroEL and its co-factor GroES) that help facilitate the correct folding of its protein products given the unusually high AT bias (88 % A+T) of its genome.This molecular dialogue was also reflected in the metabolomic analysis from bacteriome and haemolymph collected from normal and symbiont-cured female tsetse.The absence of Wigglesworthia disrupted multiple host metabolic pathways, including those of carbohydrates and amino acids, and affected downstream nucleotide biosynthesis and metabolism and biosynthesis of S-adenosyl methionine (SAM), an essential cofactor [10].

Future opportunities:
The conspicuously intimate nature of tsetse's relationship with Wigglesworthia has heretofore prevented the cultivation of this bacterium outside of the fly.This obstacle has impeded our ability to identify and characterize in more depth the specific Wigglesworthia-derived molecules that enhance tsetse fitness.However, the fact that Wigglesworthia resides extracellularly in tsetse milk provides promise that the bacterium can be grown in vitro in a biochemically similar medium.Doing so will facilitate the ability to interfere with Wigglesworthia gene expression as a means of identifying fecundityenhancing and immunostimulatory molecules that may be targetable in novel control strategies aimed at reducing tsetse population size and trypanosome vector competence.

OPEN QUESTIONS
(1) What are the Wigglesworthia-derived factors that contribute to tsetse immune system development, and how do these factors function in this capacity?(2) What are the mechanisms that facilitate metabolite exchange and regulate coordination between Wigglesworthia, its tsetse host and other members of the fly's microbiota?(3) How can we develop a medium in which we can cultivate (and potentially genetically modify) Wigglesworthia outside of its tsetse host?(4) How can Wigglesworthia be experimentally exploited to reduce tsetse's competence as a vector of pathogenic African trypanosomes?